Immunoassay method and kit to be used therein

ABSTRACT

Using two types of antibodies, i.e., a first antibody having a higher affinity for a target substance than for a competitive substance and a second antibody having a higher affinity for the competitive substance than for the target substance, a specimen is treated with these two antibodies. Then, the competitive substance in the specimen first binds to the second antibody and thus the ratio of the target substance to the competitive substance in the specimen is enlarged. As a result, the target substance becomes liable to bind to the first antibody and, in its turn, the reactivity of the target substance is elevated compared with the case of using the first antibody alone. Thus, the target substance in the specimen can be accurately assayed while avoiding the effects of the competitive substance contained in the specimen.

TECHNICAL FIELD

The present invention relates to an immunoassay method that permitsaccurate assay of a target substance in a specimen by the use of twotypes of characteristic antibodies, and a kit to be used in the method.More particularly, the present invention relates to an immunoassaymethod in which using two types of antibodies, i.e., a first antibodyhaving a higher affinity for a target substance than for a competitivesubstance and a second antibody having a higher affinity for thecompetitive substance than for the target substance, a specimen istreated with these two antibodies, whereupon the competitive substancein the specimen first binds to the second antibody and thus the ratio ofthe target substance to the competitive substance in the specimen isenlarged, so that the target substance becomes liable to bind to thefirst antibody, and as a result, the reactivity of the target substanceis elevated compared with the case of using the first antibody alone, sothat the target substance can be accurately assayed; and a kit to beused in this method.

BACKGROUND ART

Various types of substances are present in a specimen containing atarget substance to be assayed, and a target substance to be assayed anda substance competitive therewith are present in a certain specimen atthe same time in some cases. For example, a large portion of tartrateresistant acid phosphatase (TRACP) in serum is considered acidphosphatase derived from osteoclast and the assay of TRACP is considereduseful as an indication for evaluating the function of osteoclast. Thus,TRACP is a substance that is gaining interest as a bone resorptionmarker (Norio Fukunaga, Toshitaka Nakamura and Toshio Matsumoto,“Osseous Metabolism Marker”, Medical Review Co., Ltd., 1995), and it hasbeen found that besides this enzyme having enzymatic activity, fragmentas degradation products of the enzyme are also present in serum (J BoneMiner Res. 15:1337-1345, 2000; and Clin Chem. 47:74-80. 2001).

By the way, acid phosphatase in serum is divided into six bands 0 to 5from the origin by polyacrylamide gel electrophoresis. Of these, acidphosphatase corresponding to the fifth band is tartrate-resistant and iscalled Band 5 tartrate resistant acid phosphatase (TRACP 5: tartrateresistant acid phosphatase 5). This acid phosphatase is further dividedby electrophoresis into 5a which has a high content of sialic acidbonded to a sugar chain and 5b which has almost no sialic acid bonded toa sugar chain. In addition, 5a is an enzyme derived from platelet or thelike and its blood level does not vary, while only the blood level of 5bvaries with bone resorption. Therefore, it is considered that 5b is themain body of tartrate resistant acid phosphatase derived fromosteoclast. Also in “Clinical Chemistry” (Clin. Chem. 47:1497. 2001), itis recommended that ACP derived from osteoclast should be abbreviated asTRACP 5b. Accordingly, also in the present specification, phosphatasethat refers to ACP derived from osteoclast and used as an indication ofbone resorption is expressed in the term “TRACP 5b”, and tartrateresistant acid phosphatase derived from osteoclast and tartrateresistant acid phosphatase 5b (TRACP 5b ) are considered synonymous witheach other. Thus, all of them are expressed in the term “TRACP 5b”.

Besides TRACP 5b, acid phosphatase derived from erythrocyte or thatderived from platelet is present as ACP in a specimen. That is, whenhemolysis is caused by collection of a specimen, acid phosphatasederived from erythrocyte is contained in the specimen. When serum isused as a specimen, platelets are destroyed during blood coagulation inserum preparation, so that acid phosphatase derived from platelet iscontained in the specimen. Therefore, conventional methods for assayingTRACP activity do not permit assay of osteoclast-specific TRACP 5b. As amodification of these assay methods, there is known a method in whichafter a pretreatment comprising incubation of a 5-fold dilution of serumat 37° C. for 1hour, the residual TRACP activity is assayed by the useof p-nitrophenylphosphoric acid (pNPP) as a substrate in the presence oftartaric acid (“Nichidai-Ishi”, 49:904-911. 1990; and Clin. Chem.33:458-462. 1987). This method permits avoidance of the influence ofacid phosphatase derived from erythrocyte but does not permit exclusionof the influence of acid phosphatase derived from platelet. In addition,as a more specific activity assay method, there is a TRACP 5b assaymethod utilizing the difference in sensitivity to fluorine between TRACP5b and tartrate resistant acid phosphatase derived from erythrocyte orplatelet (JP-A-10-37198). This method, however, does not permitexclusion of the influence of TRACP 5a though it permits avoidance ofthe influence of tartrate resistant acid phosphatase derived fromerythrocyte or platelet. Moreover, this method is desired to be furtherimproved in precision because TRACP 5b activity is assayed in thismethod by subtracting activity not inhibited in the presence of fluorinefrom the total tartrate resistant acid phosphatase activity.

In immunoassay methods such as the method using a polyclonal antibody ofMarius E, Sari L, et al. (J Clin Endocrinol Metab. 71:442-451. 1990; andClin Chem. 46:1751-1754. 2000) and the method using a monoclonalantibody of Jussi M. Halleen, Heather Bull, et al. (J Bone Miner Res.13:683-687. 1998; Immunol Lett. 70:143-149. 1999; J Bone Miner Res.14:464-469. 1999; Clin Chem. 45:2150-2157. 1999; and Clin Chem.46:1751-1754. 2000), activity corresponding to the whole of Band 5 isassayed, so that the influence of TRACP 5a is not negligible. Inaddition, the method of Halleen, et al., which is designed to assayTRACP 5b specifically, is more specific for TRACP 5b activity derivedfrom osteoclast but indicates only slight difference between a specimenfrom a healthy person and a specimen from a patient sufferingexacerbated bone resorption. Thus, the sensitivity of TRACP 5b as a boneresorption marker is not sufficient. The low specificity for TRACP 5b ofheretofore-prepared polyclonal antibodies and monoclonal antibodies isconsidered as a cause for the defects described above. Since it has beenreported that TRACP contains fragments with no enzymatic activity in alarge amount of 10 times the amount of the active enzyme in serum, it isalso conjectured that the fragments compete with the intact enzyme inserum, so that the specificity has been low because of a disadvantageousreaction system.

Even in such a case, quantification has heretofore been carried out byan immunoassay method or the like by utilizing an antigen common to thecomplete substance with enzymatic activity, the intact enzyme, andenzyme degradation products. However, as a result, when an epitope islimited as in the case of a monoclonal antibody, measurable fragmentsand non-measurable fragments appear owing to degradation. Therefore, itcan be speculated that there is, for example, the following problem:even when one and the same target substance is assayed, there is nocorrelation among the assay results obtained by the use of various kits.In addition, it has also been reported that in the case of TRACP,examples of which have been previously given, the amount of itsfragments does not clinically reflect bone resorption (J. Bone Miner.Res., 15:1337-1345, 2000; and Clin. Chem. 47:74-80, 2001). This isconsidered as a proper example of necessity for the measurement of onlythe enzyme active form. In such a case, only the enzyme level should bemeasured by removing the fragments. It is conjectured that in general,the assay of TRACP by an activity measurement method has been notaccurate because the inactive fragments compete with the enzyme.Moreover, there has been no fragment-specific antibody for clearseparation of protein fragments, so that no means for the separation hasbeen present in itself.

DISCLOSURE OF THE INVENTION

Accurate assay of TRACP explained in the present specification has beenimpossible for a long period of time though it has been said to have aclinical significance. As reasons for this, the following two reasonsare thought of. The first reason is that there has been no antibodyhaving a high specificity for TRACP 5b indicating the activity ofosteoclast. The second reason is that protein fragments formed by TRACPenzyme degradation are present in serum in an amount larger than that ofthe active enzyme.

Therefore, in the present invention, by developing a first antibodyhaving a high binding constant value for TRACP active enzyme and asecond antibody which hardly reacts with the enzyme and binds toinactive degradation products of the enzyme and utilizing them in oneand the same reaction system, only the enzymatic activity level isaccurately assayed while avoiding the effects of the degradationproducts.

In view of such a problem, the present invention provides an immunoassaymethod in which a first antibody highly reactive with a target substanceto be assayed and a second antibody highly reactive with a competitivesubstance such as an inactive fragment are used in combination at firstto exclude the effects of the competitive substance in the reactionsystem, whereby the target substance such as an active enzyme is assayedspecifically and accurately.

That is, the present invention provides an immunoassay method in which atarget substance in a specimen is assayed by the use of two types ofantibodies, and which comprises

using the two types of the antibodies, i.e., a first antibody and asecond antibody which have the following properties: (i) the firstantibody has affinity for the target substance and a competitivesubstance, (ii) the first antibody has a higher affinity for the targetsubstance than for the competitive substance, (iii) the second antibodyhas a higher affinity for the competitive substance than for the targetsubstance, and (iv) the affinity for the competitive substance of thesecond antibody is higher than the affinity for the target substance ofthe first antibody,

bonding the target substance and the competitive substance in thespecimen to the first antibody and second antibody adsorbed on acarrier, and then

measuring the level of the bonded target substance to assay the targetsubstance in said specimen.

Furthermore, the present invention provides a kit for immunoassay of atarget substance in a specimen by the use of two types of antibodies,which comprises

the two types of the antibodies, i.e., a first antibody and a secondantibody which have the following properties: (i) the first antibody hasaffinity for the target substance and a competitive substance, (ii) thefirst antibody has a higher affinity for the target substance than forthe competitive substance, (iii) the second antibody has a higheraffinity for the competitive substance than for the target substance,and (iv) the affinity for the competitive substance of the secondantibody is higher than the affinity for the target substance of thefirst antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simple diagram showing the results of analysis using ProteinChip System of Cyphergen Biosystems. Inc. From the graphs (1) and (2),it was found that in the case of antibody Trk49, there were four kindsof fragments specifically reactive therewith in the serum of an infant.On the other hand, from the graphs (3) and (4), it was found that in thecase of antibody Trk62, there was almost no fragment specificallyreactive therewith.

FIG. 2 shows the result of assaying the sera of patients withosteoporosis before and after hormonotherapy. The two peaks observedbefore this treatment are not present after the treatment. Also fromthis fact, it is conjectured that fragments corresponding to the twopeaks are specific for bone resorption.

FIG. 3 shows the result of ELISA using the same concentration ofantibodies Trk49 and Trk62. Antibody Trk49 exhibited only lowreactivity. However, an improved reactivity was exhibited on a plate onwhich antibodies Trk49 and Trk62 had been used at the same time, ascompared with the case of using antibody Trk62 alone.

FIG. 4 shows the result of ELISA carried out by adsorbing antibody Trk62on a plate and adsorbing either antibody Trk62 or Trk49 on latexparticles. In the case of the resulting Trk62 antibody-sensitized latexreagent competitive with the plate, coloration was reduced with anincrease of the concentration of the latex reagent added. However, bycontrast, in the case of the Trk49-sensitized latex reagent, thecoloration value of an enzyme was increased in 0.1% to 0.15%experimental groups. Accordingly, it was found that the Trk49antibody-sensitized latex reagent was effective when used in combinationwith antibody Trk62 adsorbed on the plate.

MODE FOR CARRYING OUT THE INVENTION

In the immunoassay method of the present invention, two types ofantibodies, i.e., a first antibody and a second antibody are used forassaying a target substance to be assayed in a specimen. The firstantibody and the second antibody have the following properties: (i) thefirst antibody has affinity for the target substance and a competitivesubstance, (ii) the first antibody has a higher affinity for the targetsubstance than for the competitive substance, (iii) the second antibodyhas a higher affinity for the competitive substance than for the targetsubstance, and (iv) the affinity for the competitive substance of thesecond antibody is higher than the affinity for the target substance ofthe first antibody. Here, the term “competitive substance” means asubstance which has antigenicity similar to that of the target substanceto be assayed and has such a property that when an antibody against thetarget substance is prepared, the competitive substance binds to thisantibody in competition with the target substance. For example, when thetarget substance is a protein or an enzyme, the competitive substanceincludes, for example, fragments of the protein or the enzyme, variantshaving an amino acid sequence formed by substitution, deletion oraddition of an amino acid residue(s) in an amino acid sequenceconstituting the protein or the enzyme, and modification productsobtained by the addition of a phosphoric ester group to the protein orthe enzyme or the change of the sugar chain portion of the protein orthe enzyme.

In the present invention, typical examples of the target substance to beassayed are intact enzymes. When the target substance is an intactenzyme, typical examples of the competitive substance are substances nothaving the enzymatic activity, such as products produced by thedegradation of the enzyme, i.e., enzyme degradation products.

Each of the first antibody and the second antibody in the presentinvention may be any of an antiserum, a polyclonal antibody and amonoclonal antibody.

The first antibody and second antibody used in the present invention maybe prepared, for example, by immunizing an animal with the targetsubstance to be assayed, as an antigen, fusing splenocytes of the animalwith myeloma cells to prepare hybridomas, and selecting two types ofmonoclonal antibodies having the following properties, as the two typesof the antibodies, i.e., the first antibody and the second antibody fromvarious monoclonal antibodies produced by the hybridomas: (i) the firstantibody has affinity for the target substance and the competitivesubstance, (ii) the first antibody has a higher affinity for the targetsubstance than for the competitive substance, (iii) the second antibodyhas a higher affinity for the competitive substance than for the targetsubstance, and (iv) the affinity for the competitive substance of thesecond antibody is higher than the affinity for the target substance ofthe first antibody. In the present invention, the second antibody may bean antibody having affinity for both the target substance and thecompetitive substance. To obtain the two types of the monoclonalantibodies satisfying the above-mentioned four conditions with respectto the target substance and the competitive substance, the following,for example, is sufficient: the binding properties for the targetsubstance and the competitive substance of the various monoclonalantibodies are measured by ELISA or the like, and on the basis of themeasurement result, the two types of the monoclonal antibodiessatisfying the above-mentioned four conditions are selected.Alternatively, it is also possible to measure the binding constants forthe target substance and the competitive substance of the variousmonoclonal antibodies according to a well-known method (“Protein•EnzymeFundamental Experimental Methods”, Nankohdo Co., Ltd.) and select thetwo types of the monoclonal antibodies on the basis of the measurementresult. In addition, the two types of the monoclonal antibodies may beselected also by preparing affinity-chromatographic columns using eachof the various monoclonal antibodies, and assaying the target substanceand the competitive substance adsorbed on the columns.

When each of the two types of the antibodies, i.e., the first antibodyand the second antibody is an antiserum or a polyclonal antibody, thefollowing, for example, is possible: an animal is immunized with thetarget substance to be assayed, under various conditions established bychanging an adjuvant or varying immunization conditions, and the bindingproperties for the target substance and the competitive substance of theantisera or polyclonal antibodies thus obtained are measured by the samemethod as in the case of the above-mentioned monoclonal antibodies, andthe two types of the antibodies satisfying the above-mentioned fourconditions are selected.

Typical examples of the two types of the antibodies used in the presentinvention are two types of monoclonal antibodies usable for assayingTRACP 5b derived from osteoclast. Two types of monoclonal antibodiesrelating to TRACP 5b and obtained according to the present invention area first monoclonal antibody which has a still higher reactivity withTRACP 5b than do monoclonal antibodies heretofore reported, and hardlyreacts with fragments of TRACP 5b, and a second monoclonal antibodywhich reacts with the inactive enzyme fragments and hardly reacts withthe active enzyme. The utilization of these two types of the antibodiesmakes possible an immunoassay method specific for TRACP 5b derived fromosteoclast which is hardly affected by TRACP 5b inactive fragments andother isozymes in blood (derived from erythrocyte, platelet, neutrophilor macrophage).

By taking the case of these monoclonal antibodies, the two types of theantibodies used in the present invention are explained below in furtherdetail.

The above-mentioned monoclonal antibodies may be obtained by usingpurified TRACP 5b derived from human osteoclast, as an immunogen.Although in the method described below, immunization is carried out bythe use of an antigen obtained from normal osteoclast, not only thisantigen but also TRACP 5b of osteoclast tumor or the like may be used asan antigen.

The above-mentioned monoclonal antibodies are produced by hybridomasobtained by immunizing an animal with purified human TRACP 5b as antigenand fusing its cells capable of producing anti-human TRACP 5b antibodywith myeloma cells.

The above-mentioned hybridomas may be obtained by the following method.That is, human TRACP 5b obtained in the manner described above is mixedwith a well-known adjuvant such as Freund's complete or incompleteadjuvant, aluminum hydroxide adjuvant, pertussis adjuvant or the like toprepare an adjuvant liquid for sensitization, and this liquid isadministered to an animal (e.g. a mouse or a rat) subcutaneously in theabdominal cavity or intravenously in the tail, in several portions atintervals of 1 to 3 weeks to immunize the animal. Although the amount ofthe antigen used for the sensitization is usually chosen in the range of1 μg to 100 mg, it is preferably about 50 μg in general. Although thenumber of immunizing operations is generally 2 to 7, various methods areknown. Subsequently, antibody-producing cells derived from the spleen orthe like are fused with cells having proliferating capability in a testtube, such as myeloma cells or the like. The antibody-producing cellsmay be obtained from a mouse, a nude mouse or a rat.

As to a method for the above-mentioned fusion, the fusion may be carriedout by the use of a poly(ethylene glycol) (PEG) by the method of Köhllerand Milstein (Nature. 256, 495. 1975) which is already per se wellknown. The fusion may be carried out also by the use of Sendai virus orby an electrofusion method.

As to a method for selecting hybridoma capable of producing an antibodycapable of recognizing human TRACP 5b, from the fused cells, theselection may be carried out as follows. That is, the hybridoma isselected from colonies formed by cells surviving in HAT medium and HTmedium in limiting dilution of the aforesaid fused cells. When anantibody against human TRACP 5b is contained in the supernatant of theculture medium for any of the colonies formed by the fused cells seededinto a 96-well plate or the like, a clone capable of producing amonoclonal antibody against human TRACP 5b may be selected by an ELISAmethod in which the supernatant is placed on an assay plate having humanTRACP 5b immobilized thereon, and after the reaction, a secondarylabeled antibody such as an anti-mouse immunoglobulin-HRP labeledantibody is reacted with the above-mentioned antibody. As the labelingsubstance of the labeled antibody, there may be used enzymes (e.g.alkaline phosphatase), fluorescent substances, radioactive substancesand the like besides HRP. Screening of specific antibodies against humanTRACP 5b may be conducted by carrying out, as a control, ELISA using anassay plate having only BSA bonded thereto as a blocking agent,simultaneously with the above-mentioned ELISA. That is, a clone isselected which is positive on the plate having human TRACP 5bimmobilized thereon and is negative in the ELISA using only BSA.

In addition, the following measurement may be carried out at the sametime: an anti-mouse immunoglobulin antibody is seeded on a microtiterplate, the supernatant of the culture medium for the fused cells isadded thereto to carry out the reaction, purified TRACP 5b is addedthereto to bond the active enzyme and form an immune complex, and theenzymatic activity thus bonded is measured by utilizing acolor-producing substrate such as pNPP.

The first monoclonal antibody used in the present invention includesmonoclonal antibodies capable of specifically recognizing human TRACP 5bwhich react with, in particular, intact human TRACP 5b, do not undergocross reaction with acid phosphatase derived from erythrocyte, platelet,neutrophil or prostate and potato ACP, and have a low reactivity withinactive fragments of human TRACP 5b. An example thereof is a monoclonalantibody produced by hybridoma Trk62 established by the presentinventor. The second monoclonal antibody used in the present inventionincludes monoclonal antibodies capable of specifically recognizing humanTRACP 5b inactive fragments, which hardly react with, in particular,active human TRACP 5b and do not undergo cross reaction with acidphosphatase derived from erythrocyte, platelet, neutrophil or prostateand potato ACP. An example thereof is a monoclonal antibody produced byhybridoma Trk49 established by the present inventor.

In particular, the first antibody used in the present invention ispreferably an antibody having a higher affinity for active human TRACP5b than for inactive human TRACP 5b fragments, for the purpose ofspecifically bonding the active enzyme. Especially preferable examplethereof is an antibody having a larger binding constant for active humanTRACP 5b than does the second antibody. As the second antibody, suitableis an antibody having a binding constant for the inactive TRACP 5bfragments which is larger than the binding constant for active humanTRACP 5b of the first antibody. As the first antibody, a monoclonalantibody produced by hybridoma Trk62 established by the present inventoris exemplified. As the second antibody, a monoclonal antibody producedby hybridoma Trk49 established by the present inventor is exemplified.The above-mentioned hybridomas Trk49 and Trk62 were deposited as followsin Patented Organism Deposition Center (IPOD), Industrial TechnologyGeneral Research Institute (Independent Administrative Corporation),Chuo-dairoku, Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture, Japan305-8566: hybridoma Trk49 was deposited as a receipt number IPOD FERMBP-8249 on Nov. 27, 2002 and hybridoma Trk62 was deposited as a receiptnumber IPOD FERM BP-7890 on Feb. 14. 2002.

Each of the above-mentioned hybridomas is cultured on a medium usuallyused for cell culture, such as α-MEM, RPMI1640, ASF, S-clone or thelike, and the monoclonal antibody may be recovered from the supernatantof the culture medium. The following is also possible: after a nudemouse, an animal from which the hybridoma has been derived, ispreviously treated with pristane, cells are intraperitoneally injectedinto the animal to cause accumulation of ascites, and the monoclonalantibody is recovered from the ascites.

As a method for recovering the monoclonal antibody from theabove-mentioned supernatant or ascites, a conventional method may beadopted. There are exemplified salting-out with ammonium sulfate, sodiumsulfate or the like, chromatography, ion exchange chromatography, andaffinity chromatography using protein A.

By the use of the monoclonal antibodies according to the presentinvention purified by the above method, TRACP 5b in a serum specimen canbe accurately assayed. The immunoassay method of the present inventionis explained below.

The immunoassay method of the present invention may be practiced, forexample, as follows. At first, the first antibody and the secondantibody are adsorbed on an insoluble solid support such as a plate, andthe immunoassay method may be practiced by the same procedure as in aconventional ELISA method except for the above adsorption. A specimen tobe subjected to assay is first added to the support having two types ofthe antibodies adsorbed thereon. In this case, a competitive substancein the specimen undergoes antigen-antibody reaction mainly with thesecond antibody adsorbed on the support because of the difference inbinding constant between the first antibody and the second antibody, andthen a target substance in the specimen advantageously undergoantigen-antibody reaction mainly with the first antibody adsorbed on thesupport. Subsequently, the solid support is washed when occasiondemands, and then the level of the target substance attached to thesolid support is measured, whereby the target substance in the specimenmay be assayed. In this case, when the target substance is an intactenzyme such as TRACP 5b, a solution of a substrate corresponding theretosuch as pNPP (p-nitrophenylphosphoric acid) is added to the solidsupport to carry out the enzyme reaction and measure the level of thetarget substance attached to the solid plate, whereby the targetsubstance in the specimen can be accurately assayed.

In the present invention, a carrier on which the first antibody isadsorbed may be an insoluble solid support and the second antibody maybe adsorbed on a carrier dispersible in a solution, such as a latex ormay be dissolved. In this case, a target substance may be assayed asfollows. At first, the carrier dispersible in a solution, such as alatex is sensitized with the second antibody or the second antibody isdissolved. On the other hand, the first antibody is adsorbed on thesolid support. Then, a solution containing the thus treated secondantibody and a specimen to be subjected to assay are added to the solidsupport, whereby a competitive substance in the specimen is subjected toantigen-antibody reaction mainly with the second antibody and the targetsubstance is subjected to antigen-antibody reaction mainly with thefirst antibody. Subsequently, the solid support is washed when occasiondemands, and then the level of the target substance attached to thesolid support is measured, whereby the target substance in the specimenmay be assayed. In this case, when the target substance is an intactenzyme such as TRACP 5b, a solution of a substrate corresponding theretosuch as pNPP (p-nitrophenylphosphoric acid) is added to the solidsupport to assay the target substance attached to the solid support,whereby the target substance in the specimen can be accurately assayed.

As explained above, a carrier on which the first antibody is adsorbed isusually an insoluble solid support. The second antibody is used afterbeing adsorbed on the insoluble solid support, or dispersed in asolution containing a latex or the like, or dissolved.

The insoluble solid support is not limited so long as it is one which isused in a solid phase immunoassay method such as ELISA. The insolublesolid support includes, for example, those made of any of polystyrenes,polypropylenes, polycarbonates, polyethylenes, nylons, polymethacrylatesand the like. The monoclonal antibody (antibodies) according to thepresent invention is directly or indirectly bonded to any of theabove-exemplified per se well-known solid supports by utilizing physicalbonding, chemical bonding or affinity. The amount of the antibody(antibodies) used for the sensitization is often in the range of 1 ng to100 mg/ml. A specimen to be subjected to assay of human TRACP 5b isadded to the monoclonal antibody (antibodies) bonded to the solidsupport by physical bonding, chemical bonding or affinity, to carry outthe reaction. After a definite period of the reaction, the solid supportis washed and a color-producing substrate is added thereto to carry outthe reaction. As the substrate, a known substrate such as pNPP may beused.

As the carrier dispersible in a solution on which the second antibody isadsorbed, latex particles, magnetic particles and lipid particles may beexemplified. In addition, there may also be used a dispersion obtainedby bonding the second antibody to a chemical synthetic polymer, anatural polymer (e.g. a dextran) or the like and dispersing the thustreated polymer in a solution. In this case, the assay of a targetsubstance on a solid plate may be made advantageous by adding the secondantibody capable of binding to a competitive substance to a buffersolution reagent in an ELISA method. The assay of a target substance maybe made advantageous by bonding the second antibody to an insolubilizedcarrier and allowing the thus treated second antibody to compete withthe first antibody adsorbed on a solid plate. In addition, the assay ofa target substance may be made advantageous by bonding the secondantibody to a chemical synthetic polymer, a natural polymer (e.g. adextran) or the like and adding the thus treated antibody to a buffersolution reagent.

The kit for practicing the immunoassay method of the present inventioncomprises two types of antibodies, i.e., the above-mentioned firstantibody and second antibody. As described above, these antibodies may,if necessary, be adsorbed on an insoluble solid support or a carrierdispersible in a solution, such as a latex. If necessary, the kit mayfurther comprises a reagent for assaying a target substance bonded tothe antibodies.

As to the present invention, as described in detail hereinafter inExample 1, TRACP 5b fragments in various sera were investigated inpractice by the protein chip technology by using monoclonal antibodiesTrk49 and Trk62 produced by the above-mentioned hybridomas Trk49 andTrk62, respectively. As a result, it was proved that fragments of TRACP5b having a molecular weight of about 5580 Da, 5795 Da, 6860 Da or 7075Da were found in the sera of infants or the sera of patients withosteoporosis, in which bone resorption was actively in progress.Therefore, it was found that these fragments of TRACP 5b are usable asmarker molecules for diagnosing bone diseases such as a bone disease inwhich bone resorption is actively in progress. Accordingly, the bonedisease may be diagnosed by detecting these marker molecules in aspecimen (e.g. serum, plasma or blood) obtained from a patient, ormeasuring the amount of these marker molecules.

All methods known at present may be adopted for detecting or quantifyingthe marker molecules. The methods include, for example, a massspectrometry method, immunoassay method, electrophoresis method, liquidchromatography method and gas chromatography method. As the massspectrometry method, a method using a laser desorption/ionization-timeof flight-mass spectrometer (LDI-TOF MS) is exemplified. As the laserdesorption/ionization-time of flight-mass spectrometer, there may beexemplified surface enhanced laser desorption/ionization-time offlight-mass spectrometers (SELDI-TOF MS method) and matrix-assistedlaser desorption/ionization-time of flight-mass spectrometers (MALDI-TOFMS method). For example, when SELDI-TOF MS method is adopted,Protein•Biology•System II•Mass•Spectrometer (Ciphergen Biosystems, Inc.)developed by Ciphergen Biosystems, Inc. may be used. This machine isbased on a protein chip technology comprising a combination of SELDI(surface enhanced laser desorption ionization) and a time-of-flight massspectrometer. The details of the machine are disclosed in InternationalPublication No. WO 01/25791 A2, JP-A-2001-281222 and the like. Usually,in SELDI-TOF MS method, a specimen is pretreated, adsorbed on a chip andthen loaded on a SELDI-TOF MS mass spectrometer. When the specimen isserum, it is preferable to remove albumin from the system by using anadsorbent for albumin or washing the system with a buffer solution untilthe possession of electric charge by albumin owing to the ion exchangechip is ceased. The protein chip used in such a method is notparticularly limited so long as it can adsorb the above-mentioned markermolecules. As the protein chip, there may be exemplified chips (referredto also as chemical chips) in which functional groups having affinityfor proteins have been modified to give hydrophobicity, ion-exchangeproperties or the like, and chips (biochemical chips) having an antibodyagainst a protein of interest immobilized thereon.

As another mass spectrometry method, a mass spectrometry method using anESI method (electrospray ionization) is exemplified. In the case of theESI method, it is often preferable to load a specimen subjected topretreatment such as protease treatment, on a mass spectrometerconnected directly to a separating means such as high performance liquidchromatography. As the immunoassay method, there may be exemplified amethod in which a heretofore known protein is assayed by preparing apolyclonal or monoclonal antibody against the marker molecules. Such animmunoassay method includes an enzyme immunoassay method (EIA method),immunoturbidimetry method (TIA method), latex immuno-agglutinationmethod (LATEX method), electrochemiluminescence method, fluorescencemethod and the like. An immuno-chromatography method and a method usingtest paper are also effective. All of these methods are generally knownto those skilled in the art and these generally known methods may beadopted as they are.

Besides the methods described above, an electrophoresis method, liquidchromatography (LC) method, gas chromatography (GC) method and the likeare exemplified. These methods are also generally known to those skilledin the art and the measurement can be carried out by adopting thesegenerally known methods as they are.

The present invention is more concretely illustrated with reference tothe following preferable working example, which should not be construedas limiting the scope of the invention.

EXAMPLE 1

(1) Selection and Preparation of an Antigen for Preparing MonoclonalAntibodies

TRACP 5b derived from human osteoclast was prepared as an antigen forpreparing anti-human TRACP monoclonal antibodies. A purification methodis described below.

After informed consent was obtained, 130 g of the caput of humanthighbone excised by a surgical operation was frozen in liquid nitrogen,crushed with a hammer, and then suspended in 200 mL of a buffer solution(50 mM Tris-HCl, 0.3M KCl, 1 mM PMSF, 1 mM EDTA·2Na, 0.1% Triton X-100,0.02% NaN₃, 1 unit/ml aprotinin, pH 7.5) containing a proteaseinhibitor, followed by homogenization in a ultrasonic homogenizer. Thehomogenate was stirred overnight at 4° C. and centrifuged at 10,000 rpmfor 20 minutes, and the supernatant was dialyzed against 10 mM Trisbuffer (pH 8.2). Thereafter, the dialyzed solution was applied to aCM-Sepharose column (Φ40 mm×40 cm) (SIGMA) and the protein adsorbed waseluted with the same Tris buffer as above containing NaCl, whileincreasing the NaCl concentration with a linear gradient (0-0.5M NaCl).Tartrate resistant acid phosphatase activity was assayed with asubstrate p-nitrophenylphosphoric acid, and fractions containing a highlevel of the activity were pooled. The solution thus obtained wasconcentrated and then dialyzed against 20 mM Tris buffer (pH 7.2)containing 0.7 M NaCl, and the dialyzed solution was applied to aSuperdex 200 column (Φ6 mm×60 cm) (Amersham Pharmacia Biotech). In thesame manner as above, tartrate resistant acid phosphatase activity infractions obtained by elution was assayed and fractions containing theactivity were pooled. The solution thus obtained was diluted 2-fold with20 mM Tris buffer (pH 7.2) and applied to a HiTrap Heparin HP column (5mL) (Amersham Pharmacia Biotech), and the protein adsorbed was elutedwith the same 20 mM Tris buffer (pH 7.4) as above containing NaCl, whileincreasing the NaCl concentration with a linear gradient (0.35-1M NaCl).Fractions containing a high tartrate resistant acid phosphatase activitywere pooled and then concentrated to obtain 0.4 mg of purified acidphosphatase derived from osteoclast.

The amount of the protein was confirmed by A₂₈₀. As to the purity,SDS-PAGE (TIFCO) was carried out, followed by silver staining. As aresult, the purity was confirmed by the appearance of a single band at amolecular weight of about 35,000. The enzyme corresponding to the singleband was considered as purified TRACP 5b and was used as an immunizingantigen.

(2) Immunization

The purified tartrate resistant acid phosphatase derived from osteoclast(TRACP 5b ) was diluted to a concentration of 250 μg/ml with 50 mMcitrate buffer (pH 5.5), and 25 μg (100 μl) of the dilution wasthoroughly mixed with 100 μl of Freund's complete adjuvant (WAKO) untilemulsification was effected. The suspension thus prepared wasintraperitoneally administered to a Balb/c female mouse aged 6 weeks(Nippon Clear Co., Ltd.) under anesthesia with diethyl ether. After 2weeks, the same amount as above of TRACP 5b (25 μg/ml) was mixed withFreund's incomplete adjuvant (WAKO). By exactly the same operation as inthe case of the Freund's complete adjuvant, emulsification was effectedto obtain a suspension and the mouse was sensitized with the suspension.Two weeks after this procedure, the same procedure as above was carriedout. For the fourth immunization, i.e., final immunization, a dilutionof TRACP 5b (25 μg/ml) with 50 mM citrate buffer (pH 5.5) was preparedand then administered to the mouse by injection into the tail vein.

(3) Establishment of Hybridomas

Three days after the final immunization, the spleen was surgicallyremoved from the mouse sensitized with TRACP 5b, under anesthesia withdiethyl ether, and was aseptically dispersed to prepare splenocytes.Fusion was carried out according to the method of Köhller and Milstein(Nature, 256, 495, 1975). The splenocytes were fused with myeloma cellsP3-X63-Ag8-U1 (P3U1) by the use of a poly(ethylene glycol) (PEG4000)(MERK). As to the fusion ratio, the number of the splenocytes was 8×10⁷,while the number of the myeloma cells P3-X63-Ag8-U1 (P3U1) was 2×10⁷.That is, the fusion ratio of the splenocytes to the myeloma cells was4:1. The fused cells were dispersed in 10% FCS (INVITROGEN) α-MEM(IRVINE) HAT (Cosmo Bio Co., Ltd.) medium, seeded into a 96-wellsmicrotiter culture plate (Sumitomo Bakelite Co., Ltd.) and then culturedunder conditions of 37° C. and 5% CO₂.

(4) Screening

After about 2 weeks, the growth of colonies was confirmed and screeningwas conducted. A method for conducting the screening is described below.

For producing a plate for the screening, TRACP 5b purified in the aboveitem (1) was dissolved in 50 mM citrate buffer and applied into a96-well plate (Nunc) in an amount of 0.5 μg/100 μl/well. The plate wasallowed to stand at 4° C. for two nights and then washed three timeswith Tris buffer containing 0.05% Tween 20. Each well was applied with200 μl of 1.5% BSA solution in order to inhibit a nonspecific reaction,and the plate was allowed to stand overnight at 4° C. After the thuscompleted plate was washed three times with Tris buffer containing 0.05%Tween 20, 100 μl of the culture supernatant was reacted in each well andthe plate was further washed. Then, HRP-labeled anti-mouseimmunoglobulin antibody (Zymed), a secondary antibody was added to carryout the reaction. After washing, 100 μl of a 3 mg/ml color-producingsolution of o-phenylenediamine (OPD) (Nakalai), a color-producingsubstrate for HRP, in citric acid was added to each well to causecoloration for a definite period. Then, 100 μl of 1N sulfuric acid wasadded to each well as a terminating solution and absorbance was measuredat a measuring wavelength of 492 nm. Clones found to be positive by theabove procedure were subjected to recloning by a limiting dilutionmethod, and the supernatants thus obtained were checked again.

(5) Confirmation of Antibodies

The reactivity of clones Trk49 and Trk62 with purified TRACP 5b wasconfirmed by ELISA to find that clones Trk49 and Trk62 reacted with theplate with high sensitivity though they were different in affinity. As aresult, clones Trk49 and Trk62 were selected as clones which hadrecognized TRACP 5b. Antibodies produced by these clones were assayed bythe use of a monoclonal antibody typing kit (Amersham Pharmacia Biotech)to obtain the results shown in Table 1 below.

TABLE 1 Characteristics of the clones Class Light chain clone Trk49 IgG1κ clone Trk62 IgG1 κ(6) Preparation and Purification of Monoclonal Antibodies

To a Balb/c female mouse aged 10 weeks (Nippon Clear Co., Ltd.) twoweeks after administration of 0.5 ml of pristane (Aldrich) to the mousewere intraperitoneally administered 1×10⁷ cells of each of thehybridomas Trk49 and Trk62 obtained in the above item (5). After about 2weeks, ascites accumulated in the abdominal cavity of the mouse wassurgically collected under anesthesia with diethyl ether. As a result ofconfirmation by the use of the ascites stepwise diluted as a sample andby the ELISA method adopted for the screening in the above item (4), itwas found that the ascites contained a high concentration of amonoclonal antibody. The ascites was treated with 40% ammonium sulfateand dialyzed against PBS, and then the monoclonal antibody was purifiedby the use of a protein G column (Amersham Pharmacia Biotech) andconfirmed by SDS-PAGE. As a result, in the case of both monoclonalantibodies Trk49 and Trk62, a single band was confirmed at a molecularweight of about 150,000 when the antibodies were in a non-reduced state,and two bands were confirmed at molecular weights of about 50,000 and25,000 when the antibodies had been reduced with mercaptoethanol. Theamount of each of the purified antibodies Trk49 and Trk62 was about 15mg or more per mouse, namely, it was sufficient for industrialutilization.

(7) Assay of Specificity

In order to investigate the specificity of monoclonal antibodies Trk49and Trk62, the following experiment was carried out by using variousisoforms (TRACP 5b, erythrocyte, platelet, PAP (SIGMA), and potato ACP(SIGMA)). A measuring method is briefly as follow.

Each of the monoclonal antibodies purified by the use of Protein G wasapplied into a solid plate (Nunc) in an amount of 2 μg/well, and theplate was allowed to stand at 4° C. for two days. The plate was washedthree times with a 20 mM Tris (pH 7.0) washing solution containing 0.05%Tween 20, and then 200 μL of 1.5% BSA Tris (pH 7.0) was added to eachwell, followed by overnight blocking at 4° C. The plate thus producedwas washed once with the same washing solution as above, and theenzymatic activity of all of the various isoforms was adjusted to 10IU/L and 100 μl of each of the solutions thus obtained was added to eachwell of the antibody-attached plate. The reaction was carried out atroom temperature for 2 hours. After completion of the reaction, theplate was washed three times with the same washing solution as above and100 μl of a substrate for acid phosphatase was added, and the reactionwas carried out at 37° C. for 1 hour. The enzyme level in each samplewas determined from the coloration of the substrate caused by the enzymecaptured by the antibody. The measurement was carried out at aphotometry wavelength of 405 nm after the addition of 50 μl of areaction-terminating solution.

As a result, monoclonal antibodies Trk49 and Trk62 were found to have avery high specificity because they reacted only with TRACP 5b and had nocross reactivity with the other isoforms.

(8) Confirmation of Fragments Utilizing a SELDI Method

In recent years, the protein chip technology comprising a combination ofsurface enhanced laser desorption ionization (SELDI) and atime-of-flight mass spectrometer has been developed by CiphergenBiosystems Inc., USA and has been begun to be clinically utilized, forexample, for the detection of a novel tumor marker. The presentinventors tried to capture TRACP 5b fragments in serum in practice byemploying said technology and utilizing the monoclonal antibodiesproduced by the established hybridomas Trk49 and Trk62.

1) Experiment Using PS20 Chips

At first, the present inventors searched for TRACP 5b or its fragmentsin serum, which were reactive with antibodies Trk49 and Trk62, by theuse of PS20 Protein Chip Arrays (Ciphergen Biosystems, Inc). The PS20chip refers to an antibody-bonding chip and has the followingcharacteristic: a reactive substance in a specimen is first bonded to anantibody bonded to a metal surface, and the substance reacted with theantibody is dissociated from the antibody with a laser and flies in avacuum, whereby the amount and molecular weight of the substance aredetermined. A method for experimental operation of the protein chiparrays is briefly described below. At first, hybridomas Trk49 and Trk62acclimatized to a synthetic medium S-clone (Sanko Junyaku Co., Ltd.)were cultured and each of the antibodies produced by them was purifiedby the use of Protein G column (Amersham Pharmacia). The concentrationof each antibody was adjusted to 500 μg/mL and 2 μL of the solution thusobtained was applied to the PS20 chip. After the reaction was carriedout at room temperature for 1 hour, 5 μL of blocking buffer (1Methanolamine PBS pH 8.0) (WAKO) was added and the reaction was carriedout at room temperature for 10 minutes. After the blocking, the chip waswashed with 8 mL of washing buffer (0.5% Triton 100 PBS) at roomtemperature for 5 minutes. This washing was repeated twice and then thechip was lightly rinsed with PBS. The vicinities of the reaction spotswere lightly wiped and 3 μL of each of four samples, i.e., the serum ofa healthy volunteer, the serum of an infant aged 12 years or less, theserum of a patient with osteoporosis before hormone substitution therapyand the serum of the patient with osteoporosis after hormonesubstitution therapy, was applied to the chip. The reaction was carriedout overnight at 4° C. After completion of the reaction, the sample wasabsorbed by Kimwipe (Jujo Kimberley) and the chip was washed with 8 mLof washing buffer (0.5% Triton 100 PBS) at room temperature for 5minutes. This washing was repeated twice and then the chip was lightlyrinsed with PBS. Drops of water were wiped from the chip with Kimwipe(Jujo Kimberley) and 0.5 μL of a saturated sinapic acid (CiphergenBiosystems Inc)/50% acetonitrile (Wako)/0.5% TFA (Wako) solution wasadded twice. The thus produced protein chip array was read withProtein•Biology•System II•Mass•Spectrometer (Ciphergen Biosystems, Inc).

2) Results of the Experiment Using PS20 Chips

Measurement data are shown in FIG. 1 and FIG. 2. In this data format,the axis of abscissa may refer to the molecular weight of a protein ineach sample detached from the PS20 protein chip and the axis of ordinatemay refer to peak height reflecting the amount of an analyte whicharrived at a detector at the aforesaid molecular weight. In this case,unit on the axis of ordinate is arbitrary units (AU). In the experimentdescribed above, the sera of infants and the sera of patients withosteoporosis before hormone substitution therapy are sera in which boneresorption is actively in progress. As a result of the experiment, apeak near 5795 Da and a peak near 6860 Da were observed in the case ofthe above two experimental groups only when the experiment was carriedout using antibody Trk49 (FIG. 1, (2) and FIG. 2, (5)). These peaks werenot observed in the case of the sera of adults in which bone resorptionwas mildly in progress (FIG. 1, (1) and (3)). In the case of the sera ofthe infants which showed the high peaks, a peak near 5580 Da and a peaknear 7075 Da can also be confirmed besides the high peaks (FIG. 1, (2)).The peaks near 5795 Da and near 6860 Da in the case of the patients withosteoporosis disappeared after the treatment by a hormone substitutiontherapy (FIG. 2, (6)). The reason why the word “near” is used forexpressing the molecular weight corresponding to each peak is that thereis usually a measurement error of 20 or less. In addition, when theexperiment was carried out using antibody Trk62, no specific fragmentwas found in sera in which bone resorption was actively in progress(FIG. 1, (3) and (4)). As a result of the experiment, the presentinventor succeeded in finding several kinds of quite novel TRACP 5bfragments which had been not known. In practice, the fragments areproteins or degradation products thereof, which are detected at about5795 Da, about 5580 Da, about 6860 Da and about 7075 Da. These fragmentsare quite novel substances and are markers capable of reflecting boneresorption or morbidity. The amount of the fragments bonded correlatesclosely with the binding constant of each antibody. Special attentionshould be paid to the reactivity of antibody Trk62. Antibody Trk62hardly reacted with fragments to which antibody Trk49 bound, namely, itwas proved that antibody Trk62 has a high specificity for the activeenzyme. By contrast, antibody Trk49 reacted very well with thefragments. From these facts, the following is conjectured: two types ofthe monoclonal antibodies used in the present invention, i.e.,antibodies Trk49 and Trk62 have a high specificity for the fragments andthe enzyme, respectively, so that in the present invention, TRACP 5b canbe assayed specifically with high sensitivity by an enzyme immunoassaymethod using the two antibodies.

(9) Measurement of the Binding Constant of the Monoclonal Antibodies

The following experiment was carried out for determining the bindingconstant for each of antigens. As to a method for the determination, thedetermination was carried out according to “Protein•Enxyme FundamentalExperimental Methods”, Nankohdo Co., Ltd. Solutions of two kinds ofantigens, i.e., (A) purified TRACP 5b and (B) TRACP 5b fragment purifiedby the use of a Trk49 affinity column (a mixture of fragments of TRACP5b, comprising at least fragments of TRACP 5b having molecular weightsof approximately 5580 Da, 5795 Da, 6860 Da and 7075 Da, respectively)were prepared so as to have a concentration of 5 μg/mL 20 mM citratebuffer. Each well of a 96-well plate (Nunc) was sensitized with 50 μL ofeach antigen solution. After overnight standing at 4° C., each well ofthe plate was washed three times with 200 μL of 20 mM Tris buffercontaining 0.05% Tween 20, and 100 μL of a blocking agent (20 mM Trisbuffer containing 1.5% BSA and 10% saccharose) was added to each well,followed by overnight standing at 4° C. Each well of the plate was againwashed three times with 200 μL of 20 mM Tris buffer containing 0.05%Tween 20, and 20 μL of each of antibody solutions obtained by stepwisedilution of each antibody to 12 concentrations (0, 0.1, 0.15, 0.20,0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.75, 1.0 μg/mL) was added to eachwell. The reaction was carried out for 2 hours under each of conditions(i) 37° C. and (ii) 4° C. After this primary reaction, each well of theplate was washed three times with 200 μL of 20 mM Tris buffer containing0.05% Tween 20, and 50 μL of a 1000-fold diluted secondary antibody(anti-mouse IgG-HRP labeled antibody: Zymed) was added to each well. Thereaction was carried out at 37° C. for 2 hours and each well of theplate was washed three times with 200 μL of 20 mM Tris buffer containing0.05% Tween 20. Then, 100 μL of a 3 mg/mL OPD solution was added to eachwell to cause coloration. The coloration was terminated with 100 μL of a0.1N sulfuric acid solution, followed by colorimetry at 492 nm.

From the results thus obtained, the amount of the monoclonal antibodybonded is determined at first as A₄₉₂ per 1M concentration under thecondition (i). For example, since A₄₉₂ was 0.250 when the amount of theantibody on the TRACP 5b plate was 0.05 μg, the amount of the antibodybonded may be determined as follows when the molecular weight of theantibody is taken as 146,000:0.25/{0.05/(0.146×10⁻⁹)}=0.731×10⁻⁹  (1)

Next, the concentration of the antibody bonded [PL] at each antibodyconcentration under the condition (ii) is determined from the valueobtained under the condition (i).[PL]=A ₄₉₂ under condition (ii)/A ₄₉₂ per 1M concentration   (2)

Then, the concentration of free antibody [PF] is determined from theconcentration of the antibody added.[PF]=the concentration of the antibody added−[PL]  (3)

[PL]/[PF] is plotted against the concentration of the antibody bondedand the binding constant (Ka) is determined from the slope (−nK) of theplot.Ka=−slope/n  (4)

As a result of the determination by the above method, it was found thatthe binding constant (ka) values of the two monoclonal antibodies Trk49and Trk62 were as shown in Table 2 below.

TABLE 2 Binding constant of the monoclonal antibodies Monoclonalantibody Binding constant Trk49 (TRACP 5b) Ka = 0.94 × 10⁹ (TRACP 5bfragment) Ka = 5.25 × 10⁹ Trk62 (TRACP 5b) Ka = 3.73 × 10⁹ (TRACP 5bfragment) Ka = 0.69 × 10⁹

When the meaning of the above result in the reaction system is figuredout, it was found that the activity of reaction, i.e., the affinity isas follows:The reactivity of Trk49 with the fragment>the reactivity of Trk62 withthe enzyme>the reactivity of Trk49 with the enzyme>the reactivity ofTrk62 with the fragment

Therefore, the following was found: since monoclonal antibody Trk49 hasa higher reactivity with the TRACP 5b fragment than with TRACP 5b itselfand moreover, this reactivity is higher as compared with the bindingconstant of monoclonal antibody Trk62 for TRACP 5b, monoclonal antibodyTrk49 first binds selectively to the TRACP 5b fragment in the reactionsystem and monoclonal antibody Trk62 can react with TRACP 5b moreadvantageously in the reaction system in which the amount of thecompetitive fragment has been reduced. This result supports the resultof the experiment using protein chips.

(10) Measurement Experiment by ELISA

Therefore, the following five kinds of plates were produced: Trk49 1μg/well/100 μL, Trk49 2 μg/well/100 μL, Trk62 1 μg/well/100 μL, Trk62 2μg/well/100 μL, and (Trk49 1 μg +Trk62 1 μg)/well/100 μL. A method forproducing the plates was the same as in the production for the assay ofspecificity of the monoclonal antibodies in the above item (7), exceptfor the following change: the volume of the sensitizing antibodysolution 100 μL, the volume of the blocking solution 100 μL, and thevolume of a sample 100 μL. As to a primary reaction, pooled serum with ahigh TRACP 5b level (containing at least fragments of TRACP 5b havingmolecular weights of approximately 5580 Da, 5795 Da, 6860 Da and 7075Da, respectively) was added as a sample to the antibody adsorbed on theplate, and the antigen-antibody reaction was carried out with shaking atroom temperature. Then, a washing procedure was carried out and 100 μLof a synthetic substrate solution was added to each well, and theenzymatic reaction was carried out at 37° C. for 1 hour. The reactionwas terminated by the addition of a 0.2 N NaOH solution, followed bycolorimetry, whereby the enzymatic activity was determined. The resultsobtained are shown in FIG. 3.

Antibody Trk49 supposed to have a low reactivity with the enzyme and ahigh reactivity with the fragments on the basis of its binding constantshad a low avidity for the enzyme and hence gave no sensitivity necessaryfor the measurement. On the other hand, antibody Trk62 supposed to havea high reactivity with the enzyme on the basis of its binding constantshowed a good linearity. Furthermore, in the case of the plate having amixture of antibody Trk49 and antibody Trk62, a longer linearity and animproved sensitivity were attained. Since the linearity and sensitivitywere hardly improved even when the amount of each antibody was increasedfrom 1 μg to 2 μg, it is conjectured that as the amount of each antibodyinvolved in the reaction, 1 μg/well was substantially sufficient. Thatis, it is conjectured that the linearity and sensitivity were improvedbecause in accordance with the theory based on the binding constantexperiment, antibody Trk49 bound to the fragments in the reaction systemsubstantially preferentially and then antibody Trk62 bound to the intactenzyme.

(11) Fragment Absorption Method Using a Latex Reagent

Using monoclonal antibody Trk49 which had been found to be able toabsorb the fragments substantially specifically and monoclonal Trk62 ascontrol, a latex was sensitized with each of these two monoclonalantibodies to prepare a latex reagent. The preparation of the latexreagent was carried out as follows.

With 2 mL of a 1% latex suspension was mixed 2 mL of a 0.1 mg/mLsolution of each of antibodies Trk49 and Trk62, and the resultingmixture was stirred for about 1 hour. After centrifugation, theprecipitate was suspended in a 1% BSA solution and the suspension wasre-stirred for about 1 hour. After re-centrifugation, the precipitatewas suspended in a PBS solution to obtain a latex reagent. This latexreagent was diluted to 8 concentrations, i.e., 0.15% and 7concentrations attained by 2-fold dilution from 0.1% (0.1, 0.05, 0.025,0.0125, 0.00625, 0.00313 and 0.00156%) to prepare 8 latex reagents intotal. Operations in an ELISA method are explained below.

At first, 50 μL of each of these latex reagents was applied to each wellof a plate seeded with antibody Trk62 (2 μg/well). Then, 100 μL ofpooled serum with a high TRACP 5b level (8U/mL) obtained from a patientfrom whom informed consent had been obtained was added to each well, andthe reaction was carried out with shaking at room temperature for 1hour. After completion of the reaction, the plate was washed three timeswith 20 mM Tris buffer (pH 7.0) containing 0.05% Tween 20 and asynthetic substrate solution was added thereto. After incubation at 37°C. for 1 hour, 50 μL of a reaction-terminating solution (a 0.2 N NaOHsolution) was added to each well, followed by measurement at 405/490 nm.The results obtained are shown in FIG. 4. In the Trk62-sensitized latexreagent addition experiment, competition with the monoclonal antibody onthe plate was caused, so that enzymatic coloration was decreaseddepending on the concentration of the latex reagent added. However, inthe case of antibody Trk49, a significantly increased enzymaticcoloration value was obtained in the 0.1˜0.15% latex reagent additionexperiment. This fact, for example, indicates that not only simultaneoussensitization of a plate with the antibodies but also such an absorptionmethod using a carrier for adsorption such as a latex are effective forthe absorption of the competitive fragments. In addition, it was alsoeffective to use a solution obtained by dissolving antibody Trk49 in asolution, instead of sensitizing a latex with antibody Trk49.

INDUSTRIAL APPLICABILITY

As explained above in detail, the immunoassay method of the presentinvention makes it possible to assay a target substance to be assayedsuch as an active enzyme specifically and accurately by using acombination of a first antibody having a high reactivity with the targetsubstance and a second antibody having a high reactivity with acompetitive substance such as an inactive fragment of the targetsubstance, to avoid the effects of the competitive substance in thereaction system.

1. An immunoassay method in which tartrate resistant acid phosphatase 5b(TRACP 5b) is assayed in a specimen containing the tartrate resistantacid phosphatase 5b (TRACP 5b) together with a competitive substancetherein, the method comprising: providing a first antibody that is Trk62produced by a hybridoma deposited under the Budapest Treaty at NationalInstitute of Advanced Industrial Science and Technology, InternationalPatent Organism Depositary, under the accession number of FERM BP-7890,and a second antibody that is Trk49 produced by a hybridoma depositedunder the Budapest Treaty at National Institute of Advanced IndustrialScience and Technology, International Patent Organism Depositary, underthe accession number of FERM BP-8249, bonding the tartrate resistantacid phosphatase 5b and the competitive substance in the specimen to thefirst antibody and second antibody, wherein the first antibody andsecond antibody are both adsorbed on a first carrier, or wherein thefirst antibody is adsorbed on a first carrier and the second antibody isadsorbed on a second carrier dispersed in a solution or is dissolved ina solution, and then measuring the level of the bonded tartrateresistant acid phosphatase 5b to assay the amount of tartrate resistantacid phosphatase 5b in said specimen, wherein the measurement of thelevel of the bonded tartrate resistant acid phosphatase 5b is themeasurement of the amount of enzymatic activity of the bonded tartrateresistant acid phosphatase 5b, and wherein the competitive substance isa substance not having enzymatic activity of tartrate resistant acidphosphatase 5b.
 2. The immunoassay method according to claim 1, whereinthe carrier on which at least the first antibody is adsorbed is aninsoluble solid support.
 3. A kit for immunoassay of a target substancein a specimen by the use of two types of antibodies, which comprises afirst antibody that is Trk62 produced by a hybridoma deposited under theBudapest Treaty at National Institute of Advanced Industrial Science andTechnology, International Patent Organism Depositary, under theaccession number of FERM BP-7890, and a second antibody that isTrk49produced by a hybridoma deposited under the Budapest Treaty atNational Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary, under the accession number ofFERM BP-8249, wherein the first antibody and the second antibody areadsorbed on a carrier, or wherein the first antibody is adsorbed on acarrier and the second antibody is adsorbed on a carrier dispersed in asolution or is dissolved in a solution.